Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Summary

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

Abstract

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

Introduction

The formation of MGCs accompanies a number of pathological states in the human body distinguished by chronic inflammation¹. Despite agreement that mononucleated macrophages fuse to form MGCs², surprisingly few studies have shown fusion in context with living specimens^3,4. This is because clean glass surfaces that are required for most imaging techniques do not promote macrophage fusion when induced by inflammatory cytokines⁵. Indeed, if clean glass is used as a substrate for macrophage fusion, then low to intermediate magnification objectives (i.e., 10 - 20X) and more than 15 h of continuous imaging are often required to observe a single fusion event.

On the other hand, fusogenic plastic surfaces (e.g., permanox) or bacteriological grade plastic promote fusion². However, imaging through plastic is problematic because the substrate is thick and scatters light. This complicates imaging since long working distance (LWD) objectives are required. However, LWD objectives usually have low light gathering capacity compared to their coverslip-corrected counterpart. Further, techniques that exploit changes in the polarity of light passing through the specimen such as differential interference contrast are impossible since plastic is birefringent. The barriers associated with the use of plastic are further underscored by the fact that it is impossible to predict where macrophage fusion/MGC formation will occur on the surface. Together, these limitations restrict the visualization of macrophage fusion to phase contrast optics, extended total imaging durations (>15 continuous hours), and low resolution.

We recently identified a highly fusogenic glass surface while conducting single-molecule super resolution microscopy with fixed macrophages/MGCs⁴. This observation was surprising because clean glass surfaces promote fusion at the very low rate of ~ 5% after 24 h in the presence of interleukin-4 (IL-4) as determined by the fusion index⁴. We found that the capacity to promote fusion was due to oleamide contamination. Adsorption of oleamide or other compounds that similarly contained long-chain hydrocarbons made the glass fusogenic. The most fusogenic compound (paraffin wax) was micropatterned, and it imparted a high degree of spatiotemporal control over macrophage fusion and a 2-fold increase in the number of fusion events observed within the same amount of time compared to permanox. These optical-quality surfaces provided the first glimpse into the morphological features and kinetics that govern the formation of MGCs in living specimens.

In this protocol we describe the fabrication of a variety of glass surfaces that can be used to monitor the formation of MGCs from living specimens. In addition, we show that these surfaces are amenable to far-field super-resolution techniques. Surface fabrication is dependent on the goal of the experiment, and each surface is described with related examples in the proceeding text.

Protocol

Procedures that utilize animals were approved by the Animal Care and Use Committees at Mayo Clinic, Janelia Research Campus, and Arizona State University.

1. Preparing Acid-cleaned Cover Glass

NOTE: Cover glass purchased from many manufacturers may not be as clean as expected. Consider routinely cleaning batches of cover glass before any procedure where microscopy is involved.

1. Purchase high stringency cover glass. Take special care to choose the correct thickness (0.15 or 0.17 mm).
   NOTE: The choice of cover glass thickness is dependent on the microscope objective and is listed directly on the objective barrel.
2. Incubate the cover glass in 12 M hydrochloric acid in a well-ventilated chemical fume hood for 1 h with sonication (42 kHz, 70 W). Repeat this step for two additional times with fresh 12 M HCl.
3. Fill a separate beaker with ultrapure water and add the cover glass. Sonicate the cover glass for 5 - 10 min in a well-ventilated chemical hood. Repeat this step ten times.
4. Incubate the cover glass in pure acetone in a well-ventilated chemical hood for 30 min with sonication. Repeat this step for two additional times.
5. Fill a separate beaker with sterile ultrapure water and add the cover glass. Sonicate the cover glass for 5 - 10 min in a well-ventilated chemical hood. Repeat this step ten times.
6. Incubate the cover glass in 100% ethyl alcohol in a chemical hood for 30 min with sonication. Repeat this step two additional times.
   NOTE: The purity of the ethyl alcohol is important. Contaminants in the form of dissolved solids will dry on the glass and can promote macrophage fusion.
7. For long-term storage, place the acid-cleaned cover glass in a container filled with pure ethyl alcohol. Alternatively, dry the cover glass with nitrogen gas and store in a vacuum desiccator.

2. Preparation of Fusogenic Optical-quality Surfaces

1. Dissolve DMSO-free high-melting point paraffin wax in ultrapure toluene.
   NOTE: Stock concentrations are made at 10 mg/mL, and are diluted 1:9 in ultrapure toluene to make a 1 mg/mL working solution.
   Caution: Toluene should be handled with care as a teratogen. Dispose of toluene according to the institutional chemical hygiene plan.
2. Apply the paraffin working solution to a dry acid-cleaned cover glass at a volume that evenly covers the glass surface. Decant excess solution and dry the cover glass with nitrogen gas or air. For bulk preparation, use a Coplin jar designed to accommodate the cover glass.
3. Polish the cover glass by 3 strokes in the x axis and next by 3 strokes in the y axis with a lint-free wipe (see Table of Materials).
4. Immediately before the experiment is conducted, wash the cover glass with sterile ultrapure water, and subsequently sterilize for 15 - 30 min with ultraviolet light in a biological safety hood. Alternatively, sterilize the cover glass by ethylene oxide or gamma irradiation.

3. Micropatterning Hydrocarbon-containing Compounds to Confine Fusion to Predetermined Regions

1. Dry the acid-cleaned cover glass and immobilize the glass on a flat surface.
2. Using forceps, carefully immerse a gold transmission electron microscopy finder grid in a working solution of the hydrocarbon compound(s). Choose a hydrocarbon compound that meets the end goal of the experiment (see Table 1). Wick away excess solution by gently tapping the grid on filter paper, and immediately place the grid in the center of the cover glass. Allow the toluene to dry for 2 min before proceeding to the next step.
3. Make sure the grid is bonded to the glass by gently inverting the glass. If the grid detaches repeat step 3.2 using a new cover glass.
   NOTE: If a plasma cleaner is not available, omit step 3.4.
4. Plasma clean the cover glass by treatment with vacuum gas plasma.
   NOTE: The finder grid acts as a mask to protect the underlying surface adsorbed with long-chain hydrocarbons from the plasma. The regions that are unprotected by the grid are rendered non-fusogenic by exposure to plasma. The amount of time the cover glass is expose to plasma should be determined empirically.
5. Using fine-tip forceps, carefully remove the grid from the glass surface to expose the micropattern.

4. Fabricating Glass Bottom Dishes

1. Drill a 6 - 10 mm circular hole in the bottom of a 35-mm plastic Petri dish using a step drill bit.
   NOTE: It is critical to use a step drill bit to create smooth edges. If edges are too rough, the cover glass will not bond flat to the bottom of the dish. Imperfectly flat surfaces make microscopy difficult.
2. Carefully mix and degas silicone elastomer according to the manufacturer's instructions (i.e., polydimethylsiloxane; PDMS).
3. Apply a thin coating of elastomer just proximate to the edge of (i.e., circumscribing) the hole.
   NOTE: The elastomer should appear as a continuous albeit thin band surrounding the hole.
4. Gently apply either the fusogenic cover glass (described in section 2), or the dry acid-cleaned cover glass (described in section 1) to the dish in order to cover the hole surrounded by elastomer. Ensure that the cover glass appears flush with the bottom of the dish and extends beyond the diameter of the hole so that a substantial portion of the glass is in contact with the plastic. Cure the elastomer by baking at 50 °C for 2 - 3 h.
5. If fusogenic glass is preferred, UV sterilize the dish and culture cells according to standard protocols. However, if a micropattern is preferred, proceed to step 3.2 for micropattern preparation (the gas plasma used during micropatterning sterilizes the dish and strongly couples PDMS to glass).

5. Collecting Thioglycollate-elicited Macrophages

1. Inject 8-week-old C57BL/6 mice with 0.5 mL of a sterile solution of 4% Brewer's thioglycollate as previously described^3,4,6.
2. Seventy-two hours later, euthanize the animal according to approved animal care and use guidelines, and collect macrophages by peritoneal lavage with ice-cold phosphate-buffered saline supplemented with 5 mM ethylenediaminetetraacetate.
3. Centrifuge the macrophages at 300 x g for 3 min and resuspend in 1 mL of DMEM:F12 supplemented with 15 mM HEPES, 10% FBS, and 1% antibiotics (culture medium).
   NOTE: The cells are subsequently counted with a Neubauer hemocytometer. Macrophages are diluted to the appropriate concentration and applied to the surfaces. The number of cells to apply to a given surface should be carefully considered by the investigator for the purpose of the experimental question. Consult known standards in the primary literature.
4. After 30 min wash the surfaces with HBSS supplemented with 0.1% BSA to remove non-adherent cells, and replace with fresh culture medium. Return the cultures to the incubator (5% CO₂ in air at 37 °C).
5. 2 h later, aspirate the medium and replace with culture medium supplemented with 10 ng/mL of IL-4. Image the cells as described elsewhere⁴.

Results

Physicochemical parameters of materials have dramatic effects on the extent of macrophage fusion^7,8,9,10. Moreover, surface contaminants are known to promote macrophage fusion¹¹. Therefore, it is important to start with clean cover glass as a negative control for macrophage fusion. When cleaned as described in protocol 1, the cover glass ...

Discussion

The need to identify and subsequently develop optical-quality glass surfaces that promote macrophage fusion stemmed from the fact that until recently no published study directly visualized macrophage fusion in the context of living specimens^3,4. This is due to the fact that fusogenic plastic surfaces that are commonly used require LWD objectives and are largely limited to phase contrast optics. These barriers were overcome by engineering an optical-quality glass ...

Materials

Name	Company	Catalog Number	Comments
Plasma cleaner	Harrick Plasma	PCD-32G
Finder grid	Electron microscopy sciences	G400F1-Au	any gold TEM grid will work
Cover glass (22x22 mm)	Thor Labs	CG15CH	use only high stringency cover glass
Paraffin wax	Sigma Aldrich	17310
Petrolatum	Sigma Aldrich	16415	must be α-tocopherol-free if substituted
Oleamide	Sigma Aldrich	O2136	prepare fresh
Isopropanol	Sigma Aldrich	278475
Toluene	Sigma Aldrich	244511
Acetone	VWR International	BDH1101
Ethanol	Electron microscopy sciences	15050	use low dissolved solids ethanol
Hydrochloric acid	Fischer Scientific	A144C-212	use to acid wash cover glass
Slyguard 184	VWR International	102092-312	mix in a 1:10 ratio and cure at 50 °C for 4 h
35 mm petri dish	Santa Cruz Biotech	sc-351864
Dumont no. 5 forceps	Electron microscopy sciences	72705	ideal for removing TEM grid in section 3.5
FBS	Atlanta Biological	S11550
DMEM:F12	Corning	10-092	contains 15 mM HEPES
Pen/Strept	Corning	30-002-Cl
HBSS	Corning	21-023
BSA solution	Sigma Aldrich	A9576	use at 0.1% in HBSS to wash non-adherent macrophages
IL-4	Genscript	Z02996	aliquot at 10 μg/mL and store at -20 °C
C57BL/6J	Jackson Laboratory	000664	use for fixed samples or techniques that do not require contrast agents
eGFP-LifeAct mice	n/a	n/a	use for live fluorescence imaging
Kimwipe	Kimberly Clark	34155	use to polish hydrocarbon adsorbed surfaces